In recent years, with the development of research on proteins, a great number of peptides having various actions have been found. With the progress of genetic recombination techniques and organic synthetic methods of peptides, it has become possible to obtain these physiologically active peptides and their structurally analogous compounds in large amounts. Many of these peptides that have special activity are extremely useful as pharmaceuticals.
Examples of such peptides include peptides that bind to erythropoietin (EPO) receptors (EPO-R). EPO is a glycoprotein hormone with 165 amino acids, 4 glycosylation sites on amino acid positions 24, 38, 83, and 126, and a molecular weight of about 34,000. It stimulates mitotic division and the differentiation of erythrocyte precursor cells and thus ensures the production of erythrocytes. EPO is essential in the process of red blood cell formation, and the hormone has potentially useful applications in both the diagnosis and the treatment of blood disorders characterized by low or defective red blood cell production. A number of peptides that interact with the EPO-R have been discovered. (See, e.g., U.S. Pat. No. 5,773,569 to Wrighton et al.; U.S. Pat. No. 5,830,851 to Wrighton et al.; and WO 01/91780 to Smith-Swintosky et al.)
However, the clearance of peptides, particularly when administered in the circulatory system, is generally very fast. Therefore, it is desirable to improve the durability of such peptides. In addition, when the peptides are obtained from different species of animals, designed by peptide protein engineering, and/or having structures different from those of the subject, there is a risk of causing serious symptoms due to the production of antibodies. Hence, it is also desirable to improve the antigenicity of such peptides. In order to use these peptides as pharmaceuticals, it is necessary to have both improved antigenicity and durability.
Chemical modification of the peptides with macromolecular compounds such as poly(ethylene glycol) has been shown to be effective to improve the antigenicity and durability of various peptides. Thus, poly(ethylene glycol) and poly(ethylene glycol) derivatives have been widely used as peptide-modifying macromolecular reagents.
In its most common form, poly(ethylene glycol) has the following structure:HO—(CH2CH2O)nCH2CH2—OH
The above polymer, alpha-, omega-dihydroxyl poly(ethylene glycol) can be represented in brief form as HO-PEG-OH where it is understood that the -PEG- symbol represents the following structural unit:—(CH2CH2O)n—CH2CH2—
Without being limited to any particular theory or mechanism of action, the long, chain-like PEG molecule or moiety is believed to be heavily hydrated and in rapid motion when in an aqueous medium. This rapid motion is believed to cause the PEG to sweep out a large volume and prevents the approach and interference of other molecules. As a result, when attached to another chemical entity (such as a peptide), PEG polymer chains can protect such chemical entity from an immune response and other clearance mechanisms. As a result, PEGylation leads to improved drug efficacy and safety by optimizing pharmacokinetics, increasing bioavailability, and decreasing immunogenicity and dosing frequency.
For example, some active derivatives of PEG have been attached to peptides, proteins and enzymes with beneficial results. PEG is soluble in organic solvents. PEG attached to enzymes can result in PEG-enzyme conjugates that are soluble and active in organic solvents. Attachment of PEG to protein can reduce the immunogenicity and rate of kidney clearance of the PEG-protein conjugate as compared to the unmodified protein, which may result in dramatically increased blood circulation lifetimes for the conjugate.
For example, covalent attachment of PEG to therapeutic proteins such as interleukins (Knauf, M. J. et al., J. Biol. Chem. 1988, 263, 15,064; Tsutsumi, Y. et al., J. Controlled Release 1995, 33, 447), interferons (Kita, Y. et al., Drug Des. Delivery 1990, 6, 157), catalase (Abuchowski, A. et al., J. Biol. Chem. 1977, 252, 3, 582), superoxide dismutase (Beauchamp, C. O. et al., Anal. Biochem. 1983, 131, 25), and adenosine deaminase (Chen, R. et al., Biochim. Biophy. Acta 1981, 660, 293), has been reported to extend their half life in vivo, and/or reduce their immunogenicity and antigenicity.
In addition, PEG attached to surfaces can reduce protein and cell adsorption to the surface and alter the electrical properties of the surface. Similarly, PEG attached to liposomes can result in a great increase in the blood circulation lifetime of these particles and thereby possibly increase their utility for drug delivery. (J. M. Harris, Ed., “Biomedical and Biotechnical Applications of Polyethylene Glycol Chemistry,” Plenum, New York, 1992).
The presence of an amino acid or peptide arm between PEG and the attached macromolecule has demonstrated several advantages due to the variability of properties that may be introduced using a suitable amino acid or peptide. Of these amino acid or peptide arms, Norleucine (Nle) is used for analytical purposes; 14C or tritium labeled Gly is used for pharmacokinetic studies; Lys is used for branching; and Met-Nle or Met-βAla is used for PEG removal by BrCN treatment (Veronese, F. M. Biomaterials, 2001, 22, 405).
Another known type of PEG derivative with amino acid arm between PEG and the attached macromolecules is characterized by two linear PEG chains linked together through two functions of a tri-functional linker while the third function is used to bind the protein. Lysine is the tri-functional amino acid linker and the two PEG chains are linked to its alpha and epsilon amino groups while the carboxylic group is activated as hydroxysuccinimidyl esters for protein binding. This PEG derivative has the advantage of a lower inactivation of the enzymes during conjugation and its “umbrella-like” structure is effective in protecting proteins from proteolysis, in the approach of antibodies and in reducing immunogenicity (Veronese, F. M. Biomaterials, 2001, 22, 405).
PEG-linker-peptide or PEG-linker-liposome are sometimes formed as undesirable by-products when part of the activating group was incorporated into the final PEG-peptide or PEG-liposome adduct. Frances et al. (Int. J. Hematol. 1998, 68, 1) disclose that such linkers can have several types of adverse effects: (1) these linkers are not necessarily immunologically inert and there is experimental evidence that such groups are responsible for immunogenicity/antigenicity of PEG proteins; (2) some linkers moieties contain labile bonds that can be cleaved enzymatically or chemically; (3) linker moieties derived from often relatively toxic activated PEGs could lead to regulatory problems; (4) certain linker group such as triazine ring could cause crosslinking.
Chemical modification of the peptides with other compounds besides PEG has been shown to be effective to improve the activity and durability of various peptides. Examples include attachment of fatty acids (Wang et al., J. Med. Chem. 2005, 48, 3328), active transport agents (e.g., cholic acid), tight junction modulators (Johnson and Quay, Expert Opin. Drug Deliv. 2005, 2, 281), peptides (e.g., polyarginine), cytotoxic agents (e.g., doxorubicin), other polymers such as hyaluronic acid or carbohydrates, and the like.
Attachment of PEG or another chemical group to, for example, peptides may be achieved through the use of linker molecules. These linker molecules may provide multiple functional ends, allowing the attachment of several molecules through the use of a single linker. However, despite the advances made in the area of, for example, PEG or other modifier peptide-based conjugates, there remains a need for novel linker molecules to provide additional methods of molecular conjugation.
The citation and/or discussion of a reference in this section, and throughout this specification, shall not be construed as an admission that such reference is prior art to the present invention.